Castration resistant prostate cancer (CRPC) is an incurable disease for which novel and effective therapies are critically needed. CRPCs remain dependent upon androgen receptor (AR) transcriptional activity for continued growth and survival. Current and investigational therapies for CRPC directly or indirectly target the ligand binding domain (LBD) of the AR, yet the transactivation and DNA binding domains (TAD and DBD, respectively) have not been exploited as therapeutic targets. To identify novel AR transcriptional inhibitors, we have designed a high throughput yeast one-hybrid drug screen (Aim 1), in which reporter gene expression is dependent upon the constitutive transcriptional activity of a deletion mutant of the AR that lacks a functional LBD. In this system, suppression of reporter gene activity is indicative of a putative AR transcriptional inhibitor that interferes wit TAD and/or DBD function. Thus, our assay will favor the identification of a desired group of compounds that target the TAD and/or DBD and offer the promise of overcoming castration resistance. The robustness of our yeast one-hybrid screening assay has been established (Z' > 0.5), and counter-screens have been designed to exclude compounds that non-specifically inhibit reporter gene activity. The AR inhibitory properties of compounds identified in our yeast one-hybrid screen will be tested in multiple AR-dependent mammalian systems (Aim 2). Next, compounds will be evaluated for in vitro anti-tumor activity (Aim 3). The most active drugs will undergo structure activity relationship (SAR) analysis followed by repeated testing of biochemical and in vitro biologic activity in a reiterative process, the goal of which will be to identify two lead optimized compounds for assessing anti- neoplastic biological activity in vivo in AR-dependent CRPC mouse models (Aim 3). We will then evaluate the underlying mechanism of anti-AR action of compounds that exhibit selective in vivo anti- tumor activity in AR-dependent CRPC models (Aim 4). Our objective subsequent to the successful execution of the proposed work is to select a lead optimized AR transcriptional inhibitor for rapid translation to early phas human trials in patients with metastatic CRPC.